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| Status |
Public on Oct 17, 2012 |
| Title |
Tinman late |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
Tinman ChIP DNA from late embryos
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| Organism |
Drosophila melanogaster |
| Characteristics |
age: 5-8h AEL
tissue: embryo
antibody: Tinman polyclonal from Yin et al. 1998
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3-5.5-h-old and 5-8-h-old Drosophila embryos were formaldehyde crosslinked and chromatin was isolated by CsCl gradient ultracentrifugation according to the detailed protocol of Li et al., 2008. The isolated chromatin was sonicated to an average size of about 600 bp. The chromatin solution was precleared by incubating with normal rabbit IgG and Protein A/G beads (Pierce). IP reactions were carried out by incubating 100 μg of chromatin with 1 μg of affinity-purified rabbit anti-Tin antibody or control IgG overnight at 4°C. The immunoprotein–chromatin complexes were captured by incubating with Protein A/G beads, followed by consecutive washes and then eluted with a buffer containing 1% SDS and 0.1 M NaHCO3 (pH 10.0) as described (Li et al., 2008). In each ChIP experiment, a portion of the chromatin solution corresponding to 1% of that used in the ChIP reaction was used as input DNA control. The IP samples along with the inputs were purified by phenol/chloroform extraction and ethanol precipitation after the protein/DNA crosslinks had been reversed by incubation at 65 °C for 6 hrs. At this step, enrichment of known Tin targets was validated using qPCR. Finally, the DNA samples were amplified using a random primer-based DNA amplification protocol (Li et al., 2008). -- Li XY, MacArthur S, Bourgon R, Nix D, Pollard DA, Iyer VN, Hechmer A, Simirenko L, Stapleton M, Luengo Hendriks CL, Chu HC, Ogawa N, Inwood W, Sementchenko V, Beaton A, Weiszmann R, Celniker SE, Knowles DW, Gingeras T, Speed TP, Eisen MB, Biggin MD. (2008) Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm. PLoS Biol 6: e27.
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| Label |
biotin
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| Label protocol |
3.0 μg of amplified DNA per sample was reduced to a size of 50-100 bp by incubation with DNase I for 5 min at 37°C. After heat inactivation of DNase I, the fragmented DNA was end-labeled with bio-11-ddATP (Perkin Elmer) using recombinant Terminal Transferase by incubation at 37°C for 2 hr.
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| Channel 2 |
| Source name |
IgG ChIP DNA from late embryos
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryo
antibody: rabbit IgG
age: 5-8h AEL
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
3-5.5-h-old and 5-8-h-old Drosophila embryos were formaldehyde crosslinked and chromatin was isolated by CsCl gradient ultracentrifugation according to the detailed protocol of Li et al., 2008. The isolated chromatin was sonicated to an average size of about 600 bp. The chromatin solution was precleared by incubating with normal rabbit IgG and Protein A/G beads (Pierce). IP reactions were carried out by incubating 100 μg of chromatin with 1 μg of affinity-purified rabbit anti-Tin antibody or control IgG overnight at 4°C. The immunoprotein–chromatin complexes were captured by incubating with Protein A/G beads, followed by consecutive washes and then eluted with a buffer containing 1% SDS and 0.1 M NaHCO3 (pH 10.0) as described (Li et al., 2008). In each ChIP experiment, a portion of the chromatin solution corresponding to 1% of that used in the ChIP reaction was used as input DNA control. The IP samples along with the inputs were purified by phenol/chloroform extraction and ethanol precipitation after the protein/DNA crosslinks had been reversed by incubation at 65 °C for 6 hrs. At this step, enrichment of known Tin targets was validated using qPCR. Finally, the DNA samples were amplified using a random primer-based DNA amplification protocol (Li et al., 2008). -- Li XY, MacArthur S, Bourgon R, Nix D, Pollard DA, Iyer VN, Hechmer A, Simirenko L, Stapleton M, Luengo Hendriks CL, Chu HC, Ogawa N, Inwood W, Sementchenko V, Beaton A, Weiszmann R, Celniker SE, Knowles DW, Gingeras T, Speed TP, Eisen MB, Biggin MD. (2008) Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm. PLoS Biol 6: e27.
|
| Label |
biotin
|
| Label protocol |
3.0 μg of amplified DNA per sample was reduced to a size of 50-100 bp by incubation with DNase I for 5 min at 37°C. After heat inactivation of DNase I, the fragmented DNA was end-labeled with bio-11-ddATP (Perkin Elmer) using recombinant Terminal Transferase by incubation at 37°C for 2 hr.
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| Hybridization protocol |
Approximately 2.7 μg of labeled DNA was hybridized to each GeneChip Drosophila Tiling 1.0R Array (Affymetrix) in 1X MES, 3 M Tetramethylammonium Chloride (TMAC), 40 pM of control oligo B2 (Affymetrix), 100 μg/ml of sonicated salmon sperm DNA and 0.02% Triton X-100 for 18 hours at 45oC with 45 rpm rotation. The hybridized arrays were processed using GeneChip Fluidics Station 450 with the EukGE-WS2v4 protocol (Affymetrix).
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| Scan protocol |
Arrays were scanned on an Affymetrix Genechip Scanner 3000-7G.
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| Description |
Tinman binding in late embryos
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| Data processing |
Sample groups were analysed in MAT (version 2.09232006.linux) with default parameters, using the .TAG files submitted with the .CEL files.
Auxiliary output files from MAT are included in this submission
This is standard MAT output as detailed in http://liulab.dfci.harvard.edu/MAT/. The .SGR files are human readable log2 enrichment ratios per chromosomal position.
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| Submission date |
Oct 16, 2012 |
| Last update date |
Oct 17, 2012 |
| Contact name |
Boris Adryan |
| E-mail(s) |
ba255@cam.ac.uk
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| Phone |
+44-1223-760209
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| URL |
http://logic.sysbiol.cam.ac.uk
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| Organization name |
University of Cambridge
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| Department |
Cambridge Systems Biology Centre
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1QR |
| Country |
United Kingdom |
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| Platform ID |
GPL5919 |
| Series (1) |
| GSE41628 |
Genome-wide Tinman binding sites in early and late Drosophila embryos |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1020360_MFRA-3-L-Tin1-021607.CEL.gz |
28.9 Mb |
(ftp)(http) |
CEL |
| GSM1020360_MFRA-4-L-Tin2-021607.CEL.gz |
29.3 Mb |
(ftp)(http) |
CEL |
| GSM1020360_MFRA-5-L-IgG1-021607.CEL.gz |
28.2 Mb |
(ftp)(http) |
CEL |
| GSM1020360_MFRA-6-L-IgG2-021607.CEL.gz |
29.1 Mb |
(ftp)(http) |
CEL |
| GSM1020360_tinman-late.Dm35b_MF_v02-3_BDGPv4h_dm3.NR.bpmap_matscore.bar.gz |
18.7 Mb |
(ftp)(http) |
BAR |
| GSM1020360_tinman-late.Dm35b_MF_v02-3_BDGPv4h_dm3.NR.bpmap_matscore.sgr.gz |
21.7 Mb |
(ftp)(http) |
SGR |
| GSM1020360_tinman-late.bed.gz |
14.5 Kb |
(ftp)(http) |
BED |
| Processed data provided as supplementary file |
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